The goal of this project is to define the cellular and molecular mechanisms by which vascular smooth muscle cells (VSMC) respond to oxidative stress. Abnormal VSMC growth is the most prominent pathologic feature of restenosis, a major complication of percutaneous transluminal coronary angioplasty (PTCA), and contributes importantly to coronary artery disease. Our preliminary data suggest that the vascular injury associated with PTCA causes oxidative stress which initiates and sustains VSMC growth. Three exciting findings from our laboratories support this theory. 1) Following oversized balloon inflation-induced porcine coronary artery injury there is increased production of oxygen radicals (O2-, H2O2, and OH-) in the vessel wall for several weeks. 2) Administration of antioxidants, either probucol or the combination of vitamins C and E, decreases the magnitude of neointimal proliferation in vivo in the porcine coronary injury model. 3) In vitro we have shown that oxygen radicals stimulate VSMC growth and activate signal events typical of VSMC mitogens. These findings suggest that oxidative stress is an important stimulus in the vessel wall for VSMC growth and vascular lesion formation. The two general hypotheses of this grant are: 1) The vascular injury produced by PTCA initiates a self-perpetuating stimulation of oxygen radical generating enzymes creating an oxidative stress; and 2) changes in cellular redox state stimulate growth-related signal transduction events. To prove these hypotheses three aims are proposed. Aim 1: Determine the time course for increases in oxygen radical production following PTCA in the pig coronary and study the effect of polyethylene glycolated superoxide dismutase (PEG-SOD) infusion on redox state and neointima formation. Using the pig injury model we will measure production of O2- over the 2 week period after PTCA during which time the neointima forms. This will be correlated with the number of VSMC present and proliferating to prove that VSMC are the cell type responsible for the oxidizing environment. Then we will manipulate the redox state by infusing PEG-SOD to determine whether it limits neointima formation. Aim 2: Characterize the mechanisms responsible for increased oxygen radical production in injured vessels. Our preliminary data suggest that the following enzyme systems cause increased oxidative stress: mitochondrial oxidative phosphorylation, NADH/NADPH oxidases, phospholipase A2 (PLA2), and xanthine oxidase. The relative roles of these enzymes will be determined by measuring their enzyme activity, protein and mRNA expression and ability to produce O2-. To prove the importance of a specific enzyme, its expression will be correlated with the presence of proliferating VSMC using immunohistochemistry, bromodeoxyuridine labeling and in situ hybridization when possible. Aim 3: Characterize the signal pathways by which oxygen radicals stimulate VSMC growth. Preliminary data indicate that oxidative stress stimulates PLA2 activity to release arachidonic acid. Arachidonic acid stimulates VSMC proto-oncogene expression and DNA synthesis. The mechanisms by which oxygen radicals stimulate secreted and cytosolic PLA2 enzymes will be investigated focusing on the role of MAP kinase using a tissue culture model. These studies should provide insight into the mechanisms by which oxidative stress stimulates VSMC growth after PTCA, and thereby enable development of new therapeutic approaches to limit restenosis and atherogenesis.